Magnetic gradients have been used to separate magnetically responsive material from non-magnetically responsive materials for hundreds of years. Historically, the major industrial application was in the field of mining, from which derive some of the oldest inventions and basic patents. Magnetics have evolved as applied to separations, as well as in numerous other well-known applications, such as the magnetic recording industry, the medical sciences, electronics and other industrial uses.
The science of separations involving the isolation of molecules has evolved independently. Separations initially involved principles of solubility, advancing to methods based on gross physical properties of molecules, such as size and shape. With the introduction of chromatography, separation techniques were developed based initially on chemical interactions which were not well defined. This approach generally led to employing surface charge density as a basis for one form of separation. These and other methods based on similar principles achieved separation typically on the basis of one property or characteristic of a molecule and it became, and in many instances still is, customary to employ several techniques sequentially in order to isolate some specific compound of interest from a complex mixture.
With the emergence of modern biological science, the principle of affinity reactions (recognition at the molecular level via molecular "locks" and "keys") became known and was embodied in technology such as immunoassay, DNA/RNA hybridization techniques and, more recently, mammalian cell isolation. In such methods where a separation is performed, means for retrieving the bound element of a binding pair reaction (lock or key) was required. Methods involving "anchoring" one element evolved from simple, physically removable surfaces to various beads which could be retrieved by centrifugation. With this evolution came the appreciation that small beads have higher surface to mass ratios and that other forces, such as magnetics, could be conveniently used for separation.
In order for magnetic particle-based separation technology to become a reality, materials had to be developed with a property commonly referred to as superparamagnetism, which is the property whereby a magnetic particle exhibits a magnetic dipole while in a magnetic field and loses the dipole upon removal of the field. This property allows such materials to be resuspendable following a magnetic collection, which is essential for most separation applications. Given the various developments which had to occur, it is only in the last three decades or so that magnetic separations have been introduced into medical science and technology as a means for selecting out of complex mixtures material to be examined or analyzed, or alternatively as a means for performing a "bound/free" separation, as in the case of immunoassay. In view of the wide-scale use of magnetics in this and other clinically relevant testing, this step is critical for almost all such applications. With the extensive use of automation, it has become clear that there is a considerable need for improvement as regards the collection of magnetic materials. The present invention addresses this need.
Immunoassays are widely used in the clinical laboratory for determining the concentration of an analyte of biological or medical significance. The principles of immunoassays are reasonably well understood. Generally speaking, there are two categories of immunoassays: competitive assays and sandwich assays. For low molecular weight analytes such as drugs or metabolites, it is customary to perform competitive immunoassays. Typically, a fixed, limited quantity of specific antibody is allowed to incubate with a known concentration of labeled analyte and test sample containing some unknown concentration of the analyte of interest. The quantity of label bound to antibody is inversely proportional to the amount of analyte in the test sample. For quantitation, it is customary to perform a bound/free separation so that labeled analyte associated with the antibody can be detected. Assays which employ such a separation are termed heterogeneous. There are numerous ways for performing the bound/free separation which include adsorbing or covalently linking specific antibody to the inside of a tube (coated tube assay) or onto beads which can either be centrifuged or separated with filters or by magnets. Typically, a separation system should have the characteristics that the separation can easily be performed, excess reagent can be removed simply and non-specifically bound analyte can be washed free of the immobilized antibody with its specifically bound, labeled analyte. For analytes which have at least two characteristic antigenic determinants, a simpler and more precise approach is to perform a sandwich immunoassay, which uses two antibodies. One of the antibodies is directed to one antigenic binding site as a capture antibody while the other antibody is directed at the second binding site as the signal generating antibody. Thus, if the capture antibody is separated from solution, or bound on some solid support, the only way in which signal generating antibody can be bound to solid support or separated from solution is via binding to analyte. The advantages of sandwich assay are that: (1) signal is directly proportional to analyte concentration on the low end of the analyte curve; (2) extreme sensitivity can be obtained on the low concentration end; (3) sandwich assays are assays of "excesses" since capture antibody and label antibody are typically in excess of analyte, and so error is mainly related to accuracy of sample input; and (4) a wide dynamic analyte detection range (as much as 4-5 logs) is possible. Sandwich assay techniques, like competitive assays, employ a wide range of systems for performing bound/free separations.
Considerable effort has been devoted to the development of assays capable of being performed as quickly and as simply as possible. Several inventions are based on the principle of covalently attaching a fixed quantity of antibody to a well-defined region on a solid support where the latter has reasonable capillary action. See, for example, U.S. Pat. Nos. 5,126,242; 4,517,288; 4,786,606; 4,774,174; 4,906,439; 5,364,796; 4,446,232; and 4,752,562. Typically in such assays, specimen and labeled analyte are placed with great precision on such a solid support so as to permit competitive binding on the bound antibodies to occur. Next, solution is added which causes unbound labeled analyte to be carried from the binding region via capillary action. If the analyte is enzyme labeled, and if the liquid employed to "chromatograph" away unbound labeled analyte contains excess substrate, then a color is developed which will be proportional to the quantity of enzyme specifically bound. Another type of assay operates on a different principle, which effects "bound/free" separation by positioning solid phase antibody in some fraction of the total volume of the system. If the fractional volume in which specific binding takes place can be partitioned from the remainder of the system, then it will be possible to quantitate bound signal in the presence of an equilibrium quantity of "free" analyte, but the amount of "free" analyte in the detection region will be reduced by the volume element of the immobilized antibody regions divided by the total volume of the system. Such assays are referred to as "curtain assays" as this large fraction of unbound analyte and signal is effectively hidden behind a curtain.
Each of the above-described analytical systems suffers from its own peculiar deficiencies. In the case where capillary action is employed to chromatograph away unbound signal, non-specific binding of signal producing agent to the matrix can result in substantial background. In the case where signal producing agent includes labeling antibody or some part thereof as in the case of sandwich assays, non-specific binding becomes a significant concern. Thus, sandwich assays where medium to high sensitivity is required cannot be performed. In the curtain type assays, there is a finite limit on the smallness of the fractional volume where antibody can be bound. Hence, free signal analyte in that region results in low-end sensitivity problems.
Perhaps the most common and most sensitive method of assay heretofore in use involves covalently immobilizing a capture agent (e.g. monoclonal antibody) a solid support, such as a microtiter well, a cup or a tube. Such coated cup assays are well-known in the art. They have been used since the 1960's with radioimmunoassays, and remain common in many of the clinical analyzers in laboratories today, such as the Amerlite, Cyber-fluor, Delfia, and the ES-300 systems. See also U.S. Pat. No. 4,376,110. Advantages of coated cup assays include that they provide a homogeneous and single layer of analyte for analysis. Although coated cup technology is currently used in various immunoassay formats, the time required for the assay components to diffuse to the coated wall is excessive. Heating and constant shaking can reduce incubation times, but sensitive assays such as TSH and CEA still require 30-60 minutes for the incubation of analyte, signal producing agent and immobilized capture antibody. Additionally, the assay is highly dependent on the manufacture of cups with an evenly distributed coating of capture antibody on the cup surface.
Another type of immunoassay involves attachment of the capture antibody to a mobile solid phase, such as latex or other polymeric microbeads, some of which are magnetically responsive. Centrifugation, settling, filtration, or magnetic means are used to accomplish the bound/free separation. See U.S. Pat. Nos. 5,242,837; 5,169,754; 4,988,618; 5,206,159; 4,343,901; and 4,267,235. In certain cases, the act of binding to the particle, introduces a change in a property of the particle, which can be detected. While the benefits of a mobile solid phase include increased surface area and therefore decreased incubation times, problems with these assays include clogging of tubing, aggregation, settling, and in the cases where centrifugation is used, extremely complicated automation procedures. Most of the magnetic particles are large (1000-5000 nm in diameter,) so the problems of clogging and settling are particularly prevalent, and must be engineered around.
Recently, a class of magnetic materials appropriately referred to as ferrofluids have been introduced into immunoassay technology. Ferrofluids are nanosized crystals or crystal clusters of magnetite which are coated with materials that act as surfactants. Historically, most surfactants were, indeed, detergents; more recently, polymers or proteins have been used in that role. Ferrofluids have a variety of unique properties, among which is that thermodynamically they act as solutes. Like lyophilic colloids, they interact strongly with solvent and exhibit a variety of most unusual phenomena. With the availability of polymer/protein coated ferrofluids and the use of appropriate coupling chemistries, immunoassays in which ferrofluids have been used to perform bound/free separations have been devised. See U.S. Pat. Nos. 4,795,698; 4,965,007; 5,283,079; and 5,238,811. Ferrofluids have a decided advantage when compared to other capture systems, particularly those that employ relatively large magnetic particles (greater than 0.5 microns), which is attributable to translational and rotational diffusion. Thus, by employing ferrofluids in immunoassays, binding reactions proceed at diffusion controlled rates and do not require the constant mixing necessary when larger particles are used.
For polymer/protein coated ferrofluids wherein the crystal core is composed of magnetite clusters, the magnetic gradient required to effect separation is inversely related to the numbers of crystals in the clusters. Typically, crystal sizes are in the range of about 4-12 nm, while after coupling of bioligand, sizes range from about 20 nm to as large as 300-400 nm. Materials synthesized from crystal clusters up to about 120 nm that are well coated with polymer/protein will exhibit colloidal stability for long periods (such materials typically show no signs of settling for up to one month, or longer). As the size decreases within the optimal range for this bioligand-coupled material, which is 60 to 150 nm, such materials become more difficult to separate magnetically. Materials in the 20 nm range are difficult to effectively separate, even in high gradient magnetic separation devices employing very fine stainless steel wires capable of generating gradients of 150-200 kGauss/cm. Materials of 40-60 nm, which appear to be composed of cores having a cluster of three to six magnetite crystals, can be effectively collected with such gradients.
The above class of materials are particularly useful in performing bioanalytical separations, due principally to the ability of such materials to diffuse and to be magnetically immobilized. Since diffusion constants are inversely related to colloid size, the smaller bioligand-coupled ferrofluids have notable advantage over larger size materials. Further, in that smaller diameter materials have greater surface area per unit mass, such materials provide additional advantages over larger size materials when used in binding reactions. For example, less material is required to be introduced into the system; i.e., the binding particles represent a smaller volume fraction. As documented in commonly owned U.S. Pat. No. 5,466,574, due to their surface-to-mass ratio, the quantities of these ferrofluid particles can be manipulated, so as to be deposited on a collection surface in a substantially uniform thickness, which may aptly be characterized as a monolayer. This property makes possible quantitative signal detection while the magnetic particles are immobilized on wires, microtiter cups, rods, sheets, or other solid supports. However, the experience gained with forming such monolayers in external collection devices of the type described in U.S. patent application Ser. No. 08/006,071, has shown that while monolayers can be regularly and reproducibly formed in the apparatus disclosed in that application, as well as related U.S. Pat. No. 5,186,827 and commonly owned U.S. patent application Ser. No. 08/424,271, these monolayers are not sufficiently stable to withstand repeated and vigorous washing without constructing wash devices which are highly controlled, insofar as the rate of addition of wash solution and shearing force of solution removal are concerned. When too vigorously washed, the shearing force of the meniscus as it travels past the monolayer distorts the monolayer such that multilayering occurs at the bottom of the cup. Additionally, prolonged exposure of the monolayer to a high gradient magnetic field which is not radially symmetrical will result in lateral movement of the particles on the collection surface resulting in clumping in the region of highest field gradient. This can occur in radial fields where relatively small inhomogeneities exist. Thus, any inhomogeneities will result in undesired clumping and resultant distortion of the monolayer. In automated magnetic separation devices in which space is limited, the constraints imposed often detract from the ability of the device to produce highly symmetrical gradients. Consequently, the accumulation of particles in a clump precludes the possibility of reading signal generated by the labeled specific receptor while the analyte is magnetically immobilized on the cup wall. Such particle clumping also causes unbound signal to be trapped, which can result in higher background signal. Resuspension is, therefore, required in the washing and signal detection stages, which is undesirable for many reasons, including, above all, that resuspension is an extra step requiring time and manipulation, which to be reproducible on an automated machine would require considerable additional engineering and programming.